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Protective and antidiabetic effects of extract from Nigella sativa on blood glucose concentrations against streptozotocin (STZ)-induced diabetic in rats: an experimental study with histopathological evaluation

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Abstract

Background

Diabetes in humans induces chronic complications such as cardiovascular damage, cataracts
and retinopathy, nephropathy and polyneuropathy. The most common animal model of human
diabetes is streptozotocin (STZ)-induced diabetes in the rat. The present study investigated
the effects of Nigella sativa hydroalcholic extract on glucose concentrations in streptozotocin (STZ) diabetic
rats.

Methods

In this study Twenty-five Wister-Albino rats (aged 8-9 weeks and weighing 200-250
g) were tested. Rats were divided into five experimental groups (control, untreated
STZ-diabetic (60 mg/kg B.W., IP), treated STZ-diabetic with hydroalcholic extract
of Nigella Sativa (NS) (5 mg/kg B.W, IP), treated STZ-diabetic with hydroalcholic extract of NS (10
mg/kg B.W., IP) and treated STZ-diabetic with hydroalcholic extract of NS (20 mg/kg
B.W., IP and 32 days were evaluated to assess its effect on fasting blood glucose
(FBG), and in different groups fasting blood glucose (FBG) and body weight (BW) were
measured in the particular days (1, 16 and 32). At the end of the study, the animals
were fasted overnight, anaesthetized with an intraperitoneal injection of sodium pentobarbital
(60 mg/kg), and sacrificed for obtaining tissues samples (liver, pancreases). The
number of islets and cells were counted and the islet diameters were determined by
calibrated micrometer. The glycogen content in the liver was examined by Periodic
Acid-Schiff (PAS) staining.

Results

Treatment with NS (5 mg/kg b.w.) markedly increased BW gain and the FBG level was
significantly (p<0.001) reduced when compared to the control. Histopathological examination
showed that the NS (5 mg/kg b.w.) partially recovered hepatic glycogen content and
protected the great deal of the pancreatic islet cells. The number of islets, cells
and islets diameter were found statistically significant when compared to the control
(p<0.01, p<0.05).

Conclusions

Higher doses of NS did not exhibit any therapeutic effect. These results showed that
hydroalcholic extract of NS at low doses has hypoglycemic effect and ameliorative
effect on regeneration of pancreatic islets and may be used as a therapeutic agent
in the management of diabetes mellitus. The hypoglycemic effect observed could be
due to amelioration of β-cell, thus leading to increased insulin levels. Consequently,
N. sativa may prove clinically useful in the treatment of diabetics and in the protection of
β-cells against streptozotocin.

Virtual slide

Keywords:

Nigella Sativa; Streptozotocin; Hypoglycemic; Rat

Background

Nigella sativa L., commonly known as black seed, belongs to the botanical family Ranunculaceae. It
has being used in countries bordering the Mediterranean Sea, Pakistan, India and Iran,
as a natural remedy for over 2000 years [1]. Black seed components display a remarkable array of biochemical, immunological and
pharmacological actions, including bronchodilatory [2], anti-inflammatory [3], antibacterial [4], hypoglycaemic [5] and immunopotentiating effects [6].

N. sativa extract has been shown to possess immunopotentiating, anti-oxidant, anti-tumoral,
and anti-diabetic properties. The oil of N. sativa exhibits analgesic and anti-inflammatory effects in rats. Most of these properties
have been attributed mainly to the quinone constituents of N. sativa, of which thymoquinone is the main active ingredient of the volatile oil isolated
from the black seeds. Thymoquinone has been shown to possess strong antioxidant properties
and to suppress the expression of inducible NO synthase in rat macrophages [7].

Many studies have also examined the anti-diabetic effect of N. sativa in diabetic animal models. Aside from the effect of its crude aqueous extract to
restore glucose homeostasis. N. sativa petroleum ether extract significantly lowered fasting plasma levels of insulin and
triglycerides and normalized HDL-cholesterol. In this latter study by Le and collaborators,
N. sativa was also shown to enhance liver cell insulin sensitivity [1,3,7].

β cell defect and insulin resistance are essential features of non-insulin-dependent
diabetes mellitus (NIDDM) and both features are the focus of intensive investigations.
In this context, plants are source of many biochemical substances that present interesting
therapeutic properties. Some plants with anti-diabetic properties have been in use
in many Middle Eastern countries as a natural remedy for diabetes in traditional medicine;
N. sativa is one of these plants. It has a great potential in the treatment of diabetic animals
because of its combined hypoglycemic [2,4,7].

In earlier experiments we have shown that streptozotocin, given at 45 days post-infection
(dpi) affected the morphology of the reproductive organs of male and female worms
and lowered the number of viable eggs in the intestine and the amount of eggs in the
feces. However, the morphological changes were caused directly by the drug [5-7].

STZ, an antibiotic produced by Streptomyces achromogenes, is the most commonly used
agent in experimental diabetes. The mechanism by which STZ destroys β-cells of the
pancreas and induces hyperglycemia is still unclear. Many actions have been attributed
to STZ that are similar to those that have been described for the diabetogenic action
of alloxan, including damage to pancreatic β-cell membranes and depletion of intracellular
nicotinamide adenine dinucleotide (NAD) in islet cells. In addition, STZ has been
shown to induce DNA strand breaks and methylation in pancreatic islet cells [8,9]. Its diabetogenic action has been ascribed to an increase in the intracellular methylation
reaction, DNA strand breaks, and the production of nitric oxide (NO) and free radicals.NO
is involved in pancreatic destruction, where the interaction between NO and ROS modulates
oxidative damage. STZ can be used to induce different types of diabetes. For example,
to produce experimental models of Type 1 diabetes, mice are treated with high doses
of STZ, which depletesb -cells [8,10,11].

It is known that people suffering from diabetes mellitus are related to higher incidence
of bacterial and fungal infections. Diabetes Mellitus is a chronic disease which affects
the metabolism of proteins, carbohydrates and lipids. The major characteristic is
hyperglycaemia as a consequence of abnormal secretion of insulin in the pancreas (type
I) or inefficient action of insulin in the target tissues (type II) [12]. Type 2 diabetes is sharply increasing globally, including in many parts of the developing
world, in major part as a consequence of the worldwide “epidemic” of obesity. For
centuries, prior to and after the discovery of insulin, medicinal plants have been
used to normalize glycemia in diabetic patients. This disorder promotes adverse effects
in all organic systems. Diabetes exerts a negative action on the neuroendocrine axis
and those effects can enhance the action of diabetes on other organs that are dependent
on the axis [12].

Diabetics and experimental animal models of diabetes exhibit high oxidative stress
due to persistent and chronic hyperglycemia, which may deplete the activity of the
anti-oxidative defense system and promote the generation of free radicals [12]. Streptozotocin (STZ) is frequently used to induce diabetes in experimental animals
through its toxic effects on pancreatic β -cell [13] and as a potential inducer of oxidative stress. It has been reported that diabetes
induced by STZ is the best characterized system of xenobiotic-induced diabetes and
the commonly used model for the screening of anti-hyperglycemic activities [14].

The present study was designed to investigate the mechanism(s) of the hypoglycaemic
effect of N. sativa hydroalcholic extract, especially with respect to hepatic gluconeogenesis, and to
investigate its possible streptozotocin effects in diabetic rats.

Methods

Experimental procedure

Plant material and extraction procedure

The NS seeds were purchased from a local herb store in Urmia, Iran. The specimens
have been kept at the Department of Pathology, Veterinary Medicine Faculty, Urmia
University, Iran. The seeds of NS were powdered by a grinder. Subsequently, 20 g of
the powdered seeds were added to 400 ml of distilled water, and the extraction was
obtained by steam distillation. The distillation process was continued until 200 ml
of distillate was collected. The distillate was extracted three times with chloroform
[15].

Chemicals and streptozotocin-induced diabetes

Streptozotocin was purchased from Sigma chemical Co. Other chemicals were purchased
from Merck Company (Germany). Diabetes mellitus was induced by single intraperitoneal
(IP) injection of freshly prepared STZ at dose of 60 mg/kg b.w. dissolved in 0.01
M citrate buffer, pH 4.5. After 24 h of STZ injection, and overnight fast, blood was
taken from tail artery of the rats. Animals with FBG level of higher than 250 mg/dl
were selected for the diabetic groups.

Animals

In current study, 25 Wister-albino rats of both sexes, weighing 160-200 g with averagely
32 days old were utilized. The animals were kept in individual propylene cages under
standard laboratory conditions by the dimensions of 30×50×25 cm3 two by two. Rats were maintained on a 12 hour light/dark cycle at 22± 1°C and 50±
10% humidity. The animals were kept in standard room conditions and fed with standard
rat diet and water ad libitum. All animals received human care according to the criteria
outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the
National Academy of Sciences and published by the National Institutes of Health.

Experimental design and sample collection

Rats were divided into five groups (n = 5 in each group). Diabetes was induced in
all groups except the control group by a single intraperitoneal (i.p.) injection of
STZ (60 mg⁄kg) freshly dissolved in 5 mmol⁄L citrate buffer (pH 4.5), as described
previously [16]. One day after STZ injection, diabetes was confirmed by measuring blood glucose levels
in blood samples from the tail vein with a One Touch Glucometer (Life scan; Johnson
& Johnso, New Brunswick, NJ, USA). Rats with blood glucose levels 250 mg ⁄dL were
considered diabetic. Detailed descriptions of the five groups are as follows: Group
A (control group), rats were injected with an equal volume of vehicle (citrate buffer);
Group B, untreated STZ-diabetic (60 mg/kg b.w., IP); Group C, treated STZ-diabetic
with hydroalcholic extract of NS (5 mg/kg b.w., IP); Group D, treated STZ-diabetic
with hydroalcholic extract of NS (10 mg/kg b.w., IP); Group E, and treated STZ-diabetic
with hydroalcholic extract of NS (20 mg/kg b.w., IP), and 32 days were evaluated to
assess its effect on fasting blood glucose (FBG), and in different groups fasting
blood glucose (FBG)and body weight (BW) were measured in the particular days (1, 16
and 32).

Histological examinations

The rats were euthanized by a lethal dose of sodium pentobarbital. The histopathological
samples (liver and pancreas) were fixed in 10% neutral buffered formalin. Fixed tissue
samples were processed routinely by the paraffin embedding technique. In Histopathology,
the number of islets and the number of islet cells of each islet were counted. The
islet diameters were measured using calibrated micrometer by taking of fixed number
of islets in all groups.

Statistical analysis

All data were expressed as mean ± SD. Statistical analysis of data for BW and BG was
performed one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for
multiple comparisons was used to compare differences among experimental groups. The
statistical analysis of data for the number of islets and the number of islet cells
and islets diameter (μm) in pancreatic tissues was done using one-way analysis of
variance (SPSS 21.0) followed by Tukey post hoc test. P values of less than 0.05 were
considered as significant.

Results

Effect of N. sativa extract on body weight

Effect of hydroalcholic extract of N. sativa on body weight in STZ-induced diabetic rats were summarized in the Table 1. The changes of rat body weights in five groups were investigated. There was a progressive
increase in the body weight in the control and a progressive decrease in the STZ group,
whereas the body weight in the STZ+ NS groups (3, 4, 5) showed a progressive decrease
(Table 1). Administration of NS attenuated the weight loss induced by STZ injection in rats.
In the present experimental condition, the treatment of STZ+ NS alone had no significant
effect on changes in body weight. The mean BW of animals in group 1, 2 and 3 at the
16th day of this study were 201, 182.4 and 192.2 respectively while at 32th day of
the study they were found to be 204.4, 168.8 and 185.2 respectively. (Group 2, 4 and
5; Table 1).

The effect of STZ in 16 days showed a gradually increasing of blood glucose level
which it was (480.6 35.3) in 2 week and it reached to (565.4 30.9) mg/dl during 32
days after injection of STZ. The mean±SE of Blood Glucose concentration level in STZ+
NS) groups (5, 10, 20 mg/kg b.w) decreased from 323.2 32.2, 513.2 42.7 and 517.6 27.3
mg/dl in the 32 days (Table 2). These results suggest that 5 mg/kg b.w. is the most effective dose for assessing
the anti-hyperglycemic potential of hydroalcholic extract of NS in diabetic rats.
However, higher doses up to 20 mg/kg b.w. did not exhibit any dose dependent effect.
Treatment with NS (5 mg/kg b.w.) markedly increased BW gain and the FBG level was
significantly (p<0.001) reduced when compared to the control.

Histopathological findings

Histopathological study of islets of Langerhans revealed that size of Langerhans islets
in diabetic control group had a significant difference in comparison with all treatment
groups (P < 0.05). There was no significant difference in size of Langerhans islets
between the treated rats (with STZ+ NS) and STZ rats.

Effect of hydroalcholic extract of N. sativa on the number of islets and the number of islet cells and islets diameter (μm) in
pancreatic tissues in rats were summarized in the Table 3. Light microscopy evaluation of the pancreas of control rats (Group 1) showed normal
pancreatic islet structure (Figure 1A). In contrast, sections of the pancreas from untreated diabetic rats (Group 2) disclosed
that the islets were comparatively small and shrunken and the islet cells were degenerated
(hydropic degeneration) and necrotized (Figure 1A) (Table 3). N. sativa treatment at dose of 5 mg/kg b.w. (Group 3) protected the great deal of the Langerhans
islet cells. Nevertheless, light hydropic degeneration and necrosis were seen in the
remaining cells (Figure 1C). N. sativa treatment at doses of 10 and 20 mg/kg b.w. did not show significant difference comparison
to untreated diabetic rats (Table 3). The histochemical PAS staining demonstrated that STZ-induced diabetes resulted
in approximately complete depletion of hepatic glycogen in comparison to the control
group (Figure 1A,B). NS treatment at dose of 5 mg/kg b.w (Group 3) partially recovered the hepatic
glycogen content (Figure 1C). Treatment with NS extract at doses of 10 and 20 mg/kg b.w. did not show remarkable
change in hepatic glycogen content (Figure 1D).

Table 3. Effect of hydroalcholic extract of N. sativa on the number of islets and the number of islet cells and islets diameter (μm) in
pancreatic tissues in rats

Figure 1.Histopathological study of islets of Langerhans and effect of hydroalcholic extract
of N. sativa on the number of islets and the number of islet cells and islets diameter (μm) in
pancreatic tissues in rats. A- Diabetic group, shrunken islets of Langerhans displaying degenerative and necrotic
changes and vacuolization in diabetic rats without treatment (H&E × 400); B- NS treated group (5 mg/kg b.w.). NS protected the majority of cells in the islet
of Langerhans. However, the remaining cells exhibit light hydropic degeneration, degranulation,
and necrosis. In addition, a few cells with a picnotic nucleus are indicated (H&E
× 400). C- Complete glycogen depletion is seen due to STZ-induced diabetes (PAS staining ×
400) and The hepatic glycogen level remarkably elevated after 32 days treatment with
NS at dose of 5 mg/kg b.w. (PAS staining × 400). D- The hepatic glycogen content did not change considerably after 32 days treatment
with NS at dose of 10 and 20 mg/kg b.w. (PAS staining × 400).

Discussion

Diabetes mellitus is a chronic, systemic, metabolic disease defined by hyperglycemia
and characterized by alterations in the metabolism of carbohydrate, protein and lipid.
Oxidative stress thought to be increased in a system where the rate of free radical
production is increased and/or the antioxidant mechanisms are impaired. In recent
years, the oxidative stress-induced free radicals have been implicated in the pathology
of insulin dependent diabetes mellitus [3,5,8-10].

In our study, a significant weight loss was observed in the diabetic group while NS
treated (5 mg/kg b.w.) rats exhibited significant increase in the BW in comparison
to diabetic group (Group 2) but was lower than in the normal controls. This effect
on the BW was not observed at higher doses of extract. This finding is in agreement
with Kanter et al., 2004 reported that NS markedly improved BW gain in STZ-induced
diabetic rats. A possible explanation for this might be that NS reduces hyperglycemia,
and therefore protein wasting due to inaccessibility of carbohydrate does not occur
[17].

In present study, the hydroalcholic extract of NS at dose of 5 mg/kg b.w. revealed
a significant hypoglycemic effect in STZ-induced diabetic rats by diminishing the
FBG levels. The FBG lowering effect of that was further increased after 32 day treatment.
In addition, results showed that the anti-hyperglycemic effect of the NS extract is
time dependent. This finding is in agreement with Fararh et al., 2002 [18].

In present study, the lowering effects of black seed oil on blood glucose were correspondent
with the previous trials. Some studies have been conducted on the characterization
of the bioactives and mechanisms mediating its anti-hyperglycemic action. In an experimental
study, Alsaif [19] reported that blood glucose lowering effect of black seed oil was due to improved
insulin insensitivity in diabetic rats. Another study proposed its hypoglycemic effect
is due to improved extrapancreatic actions of insulin rather than by stimulated insulin
release [20]. Furthermore, Abdelmeguid et al. [21] reported that the anti-hyperglycemic effect of black seed oil and its active component
thymoquinone could be due to reduction of oxidative stress, thus preserving pancreatic
β -cell integrity lead to insulin levels increase. Furthermore, the black seed oil
contains many bioactive constituents such as thymoquinone, p-cymene, pinene, dithymoquinone
and thymohydroquinone [22].

The increase in glycogen levels could be due to the antidiabetic activity of NS, streptozotocin
induces degeneration of the pancreas with a lobular atrophy and a decline in size
and number of Langerhans islets [23,24]. In this study, the damage of pancreas in STZ treated diabetic rats and regeneration
of Langerhans islets by NS extract was observed. Furthermore, the number of islets,
islet cells and islets diameter significantly increased in NS (5 mg/kg b.w.) treated
group compared to STZ-induced diabetic group. However, there have been no morphometric
studies to date examining the pancreatic structure in STZ-diabetic rats treated with
NS extract. Histopathlogically, treatment with the hydroalcholic extract of NS (5
mg/kg b.w.) revealed partial regeneration of the islet cells with light hydropic degeneration
and necrosis in the remaining cells. These findings are in accordance with the results
reported by Kanter et al. [17].

Measurement of the effect of N. sativa on gluconeogenesis and liver glucose production helps to clarify part of the hypoglycemic
mechanism since hepatic glucose production through gluconeogenesis is known to contribute
to hyperglycemia in diabetic patients. Research on isolated hepatic cells showed a
significant decrease in glucose production from gluconeogenic elements like glycerol,
alanine and lactate in Nigella sativa oil-treated animals as compared to the untreated animals [3-5]. This significant decrease in liver glucose output and ameliorative effect on regeneration
of pancreatic islets suggests that the observed antidiabetic action of N. sativa is at least partially mediated through an effect on hepatic gluconeogenesis.

Conclusions

In conclusion, based on the experimental findings, it was suggested that administration
of N. sativa, at a safe dose level, suppresses STZ-induced diabetic in the rat. We believe that
further preclinical research into the utility of N. sativa treatment may indicate its usefulness as a potential treatment in diabetic patients,
our results suggested that hydroalcholic extract of NS at low doses has beneficial
effect on FBG level and ameliorative effect on regeneration of pancreatic islets and
may be used as a therapeutic agent in the management of diabetes mellitus.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

SA and RH participated in the histopathological evaluation, performed the literature
review, acquired photomicrographs and drafted the manuscript and gave the final histopathological
diagnosis and designed and carried out all the experiments. JJ is the principal investigator
of the laboratory in which the research was performed and contributed to the interpretation
of the data and writing of the manuscript. DKH, RM, FKH, MT and HA edited the manuscript
and made required changes and wrote the manuscript. All authors have read and approved
the final manuscript.

Acknowledgments

The authors are deeply grateful to Department of Pathology, Urmia University, Urmia,
Iran, for their excellent technical assistance in preparing the histological specimen.